JPH0820443B2 - How to make a stratum corneum specimen - Google Patents

Description

TECHNICAL FIELD The present invention relates to a sample preparation method for forming a stratum corneum of a skin into a sample, which includes cosmetics, foods, pharmaceuticals,
It can be used in fields such as skin quality inspection.

[Conventional technology]

Originally, when selling articles such as cosmetics that are required to keep the skin in good condition, it is necessary to accurately grasp the skin condition of the user and instruct makeup accordingly. Based on such an idea, in recent years, it has become an important subject to know the skin quality, skin property, skin condition, etc. by observing or measuring the skin condition.

Conventionally, as a method for observing these skin conditions, many methods have been proposed, but by observing the stratum corneum of the skin surface in detail, an accurate skin inspection can be performed,
I understand. Conventionally, as a method of examining the surface condition of the skin, (a) a method of directly observing the skin with the naked eye.

(B) Sump method (a method of dripping an organic solvent such as amyl acetate onto a celluloid plate to bring the celluloid plate into close contact with the skin, and then peeling the celluloid plate to prepare a skin replica, and observing the skin replica).

(C) A method of displaying a magnified image of the skin on a monitor and observing the image using a device such as a videoscope. And so on.

[Problems to be solved by the invention]

However, the method (a) or (c) is based on the appearance findings and is convenient for the measurement of the skin gloss and the like, but the condition of the skin could not be precisely investigated. Further, the method (b) is an advantageous method for examining the roughness and unevenness of the skin, but it merely measures the surface shape. Therefore, in order to investigate the skin condition in more detail,
The best way is to collect the stratum corneum and observe it.

As a method for preparing this stratum corneum sample, for example, a known adhesive is applied to a transparent plate, and after it is brought into close contact with the skin, it is peeled off, the corneum layer is retained on the transparent plate, and this is directly used as the stratum corneum sample. A method can be considered. However, according to this method, a clear and well-preserved stratum corneum sample cannot be obtained, and the skin of the subject is often damaged.

Therefore, it is difficult to precisely observe or inspect the condition of the skin.

[Means for solving problems]

The present invention has been made in view of the above-mentioned matters, and a sticky substance soluble in an organic solvent is applied, and a tape having a skin stratum corneum adhered to the sticky substance is applied with a fixing agent in advance. By sticking it on a transparent plate and then removing this adhesive tape using an organic solvent, the stratum corneum is fixed to the transparent plate, and if necessary, this is dyed to create a stratum corneum specimen. is there.

[Action]

According to the method of the present invention, by taking the above-mentioned means, the stratum corneum can be collected from the skin with a tape coated with an adhesive substance soluble in an organic solvent. And, by the above means, a clear and well-preserved stratum corneum sample can be obtained, and if the stratum corneum cells are observed and measured using this stratum corneum sample, as a result, the skin condition can be precisely and quickly investigated. You can

The stratum corneum sample provided by the present invention is a kind of the stratum corneum which is a thin-film-like group of cells existing in the outermost layer part of the epidermis which mainly covers the surface of human skin and is fixed on a transparent plate. It is a tissue sample. The above-mentioned stratum corneum specimen is capable of observing the stratum corneum cells in detail by staining or the like, and is particularly suitable for microscopic observation. When there is a nucleus in the stained stratum corneum cells, the nucleus can be clearly observed, and the keratinized state of the epidermal cells can be understood, so it is also possible to know the metabolic rate of the epidermal cells and the state of rough skin. is there.
In addition, by measuring the area, circumference, flattening ratio, homogeneity, etc. of the stratum corneum cells, it is possible to objectively evaluate the degree of skin aging and skin properties. Of course, it is also preferable to project a magnified view on the screen of the above stratum corneum sample using a device such as a videoscope, and observe and measure this.

Next, in the method for creating a stratum corneum sample according to the present invention,
The adhesive substance used is a resin or polymer compound that is soluble in an organic solvent, and specific examples include polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal,
Polyvinyl chloride, polyvinylidene chloride, polyvinyl ether, polybutene, asphalt, and natural rubber or its derivatives, SBR rubber, NBR rubber, butadiene-vinyl pyridine rubber, butyl rubber, chloroprene rubber,
Recycled rubber, cyanoacrylate rubber, gum arabic,
Rubber materials such as tragacanth rubber are listed, and these 1
One kind or a mixture of two or more kinds is used alone or in another base material. Among the above-mentioned adhesive substances, natural rubber or its derivative, SBR rubber, NBR rubber, butadiene-vinylpyridine rubber, butyl rubber, chloroprene rubber, recycled rubber, cyanoacrylate rubber, gum arabic, tragacanth rubber, etc. The rubber substance is preferably used in the method of the present invention in that it is detachable when the tape is removed and does not change the stratum corneum cells. Of course, in the method of the present invention, a tape coated with the above-mentioned adhesive substance, such as a commercially available cellophane tape, is also preferably used. However, a tape using a gum substance such as a gum tape as an adhesive substance cannot be used in the method of the present invention because the gum substance is insoluble in the above-mentioned organic solvent. It should be noted that the material of the above-mentioned tape itself may be one that is generally or industrially used, and one having a uniform thickness, one having pores, one having a corrugated shape, etc. can also be used. .

The organic solvent used is one that can dissolve the sticky substance, and specific examples of such an organic solvent include methanol, ethanol, propanol, butanol, ether, ascent, chloroform,
Hexane, THF, ethyl acetate, o-xylene, m-xylene, p-xylene, methylbenzene, ethylbenzene, propylbenzene, benzene, toluene, amyl acetate and the like can be mentioned, and one or more of these can be mixed or used together. To be used.

Next, the fixing agent used in the method of the present invention is one that fixes the stratum corneum to the transparent plate, does not denature the stratum corneum cells, and is not readily soluble in the above organic solvent. Examples of such an adhesive include known adhesives, proteins and the like, but known adhesives and the like are preferably used because the obtained stratum corneum sample is clearer, and specific examples include: Polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride,
Polyvinylidene chloride, polyvinyl ether and other polyvinyl adhesives, melamine resin adhesives, phenol resin adhesives, resorcinol adhesives, xylene resin adhesives, furan resin adhesives, polyisocyanate resin adhesives, polyester resin adhesives, polyamides Adhesive, polybutene adhesive, ethylene copolymer adhesive, thermoplastic polyester adhesive, polyester acrylate adhesive, silicone rubber adhesive, phenol mixed adhesive, epoxy adhesive, cyanoacrylate adhesive, anaerobic Polyester Adhesive, Polybenzimidazole Adhesive, Polyimide Adhesive, Polyimide Amide Adhesive, Trenees Adhesive, Metalloxane Adhesive, glue Adhesive, Casein Adhesive, Cellulose Adhesive, Viscose System adhesives It is to use a mixture of one or more of these. Among these adhesives, the polyvinyl-based adhesive is most preferable because it is difficult to dye when dyed.

Examples of the above proteins include albumin such as ovalbumin, milk albumin, myogen, serum albumin, globin, leucosine, serum globulin, β-lactoglobulin, lysozyme, myosin, edestin, insulin, fibrinogen, thyroglobulin, etc. Globulin, soluble collagen, elastin, keratin, silk fibroin, spongin, gorgonin, histone, protamine, egg white, gelatin, peptone, peptide, other, phosphoprotein, lipoprotein, glycoprotein, etc., and one of these or A mixture containing two or more kinds alone or contained in another base material such as a polyhydric alcohol is used.

Further, in the method of the present invention, the transparent plate used is transparent, does not interfere with observation of the stratum corneum specimen, and is insoluble or hardly soluble in an organic solvent. Specific examples thereof include glass plates or resins such as polymethylpentene and polyethylene. Among them, glass plates are most preferably used from the viewpoint of transparency and solvent resistance.

In the method of the present invention, first, the tape coated with the above-mentioned adhesive substance is brought into close contact with the skin, and then the tape is peeled off to collect a part of the stratum corneum on the tape. Next, this tape is attached to a transparent plate coated with the above-mentioned adhesive in advance. Here, it is important to apply the adhesive agent to the transparent plate in advance.If this pretreatment is not performed, the stratum corneum is separated from the transparent plate in the steps such as tape peeling described below. Therefore, it is inappropriate. When a tape holding a stratum corneum is attached to a transparent plate, the tape width is 3 to 10 mm.
It is preferably about the same. If the width of the tape is too wide, the permeability of the organic solvent is poor and it is inconvenient to handle. Next, the transparent plate to which the above-mentioned tape was attached was immersed in the above-mentioned organic solvent for 1 to 3 hours to dissolve or swell a part of the adhesive substance adhering to the tape to enhance the peelability of the tape. After that, carefully peel it off from the transparent plate. further,
Subsequent immersion in an organic solvent is also suitable for the purpose of removing the remaining sticky substance. As described above, the method of the present invention is characterized in that the stratum corneum collected on the tape is transferred to the transparent plate and used as a sample. Then, after transferring the stratum corneum to the transparent plate, operations such as dyeing and balsam encapsulation are usually performed by a conventional method to obtain a stratum corneum sample.

〔Example〕

 Next, examples of the method of the present invention will be described.

<Example-1> A cellophane tape having a width of 12 mm and a length of 20 mm (commercially available adhesive cellophane tape is cut and used) is stuck to the cheek, and after pressing for a while, gently peel it off and use scissors. The width is about 6 mm and the length is 20 mm, and this is attached to a slide glass on which a polyvinyl acetate adhesive has been uniformly applied.

Next, this slide glass is immersed in xylene for 2 hours. Next, the tape is gently peeled off using tweezers, and then dipped in xylene for another hour.

Then, after taking this out and drying the xylene, a gentian violet-brilliant green dyeing solution (gentian violet 1 g and brilliant green
It was immersed in 0.5 g of distilled water (100 g) for 5 to 10 minutes, washed with water, dried, and enclosed in balsam by a conventional method to obtain a stratum corneum sample.

The obtained stratum corneum specimen is a clear stratum corneum specimen which has never been seen before, and as shown in FIG. 1, the stratum corneum cells are clearly observed, and particularly the normal or abnormal state of keratinization can be observed in detail. Therefore, the metabolism and aging degree of the skin, the skin condition, etc. can be examined.

<Example-2> A cellophane tape having a width of 12 mm and a length of 20 mm (commercially available adhesive cellophane tape is cut and used) is attached to the cheek, and after pressing for a while, gently peel it off and use scissors. The width is about 6 mm and the length is 20 mm, and this is attached to a slide glass on which a polyvinyl acetate adhesive has been uniformly applied.

Next, after immersing this slide glass in xylene for 2 hours, the tape is gently peeled off using tweezers, and then further immersed in xylene for 1 hour. Next, after taking this out and drying the xylene, it was immersed in a gentian violet-brilliant green dyeing solution (gentian violet 1 g and brilliant green 0.5 g dissolved in 100 g of distilled water) for 5 to 10 minutes, washed with water and dried. rear,
A horny layer sample was obtained by encapsulating balsam by a conventional method.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-1, skin metabolism and aging degree,
I was able to check the skin condition.

<Example-3> A cellophane tape having a width of 12 mm and a length of 20 mm (commercially available adhesive cellophane tape is cut and used) is stuck to the cheek, and after pressing for a while, gently peel it off and use scissors. The width is about 6 mm and the length is 20 mm, and the mixture is gently attached to a slide glass on which the mixture of ovalbumin: glycerin = 1: 1 by weight is evenly applied, and left at a temperature of 60 ° C. for 2 hours.

Next, this slide glass is immersed in ethanol for 10 minutes, dried, and further immersed in xylene for 2 hours.
Gently remove the tape with tweezers and then soak in xylene for an additional hour. Next, after taking this out and drying the xylene, a gentian violet-brilliant green dyeing solution (gentian violet 1 g
And 0.5 g of Brilliant Green was dissolved in 100 g of distilled water) for 5 to 10 minutes, washed with water, dried and sealed in balsam by a conventional method to obtain a stratum corneum sample.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-1, skin metabolism and aging degree,
I was able to check the skin condition.

<Example-4> A paper tape having a width of 10 mm and a length of 20 mm was uniformly coated with an adhesive substance containing gum arabic, tragacanth or the like, and the adhesive tape was stuck to the cheek, pressed for a while, and then gently peeled off. Use scissors to make the width about 6 mm and the length 20 mm, and attach it to the slide glass on which the cellulosic adhesive has been uniformly applied.

Next, this slide glass is immersed in acetone for 2 hours, then the tape is gently removed using tweezers, and then further immersed in xylene for 1 hour. Next, this was taken out, xylene was dried, hematoxylin-eosin staining was performed, and after drying, it was enclosed in balsam by a conventional method to obtain a stratum corneum sample.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-1, skin metabolism and aging degree,
I was able to check the skin condition.

<Example-5> An adhesive tape obtained by uniformly applying an adhesive substance containing natural rubber to a plastic tape having a width of 10 mm and a length of 20 mm was attached to the inner side of the upper arm, and gently peeled off. The same treatment as in 1 was performed, and Giemsa staining was performed to obtain a stratum corneum specimen.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-1, skin metabolism and aging degree,
I was able to check the skin condition.

<Example-6> Cellophane tape having a uniform distribution of pores and having a width of 10 mm and a length of 20 mm (commercially available adhesive cellophane tape is processed and used) is attached to the outer side of the upper arm and gently peeled off Then, the same treatment as in Example-2 was performed to obtain a stratum corneum sample.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-2, the metabolism and aging degree of the skin,
I was able to check the skin condition.

〔The invention's effect〕

According to the present invention, by configuring as described above, it is possible to prepare a specimen that is clear, easy to observe, and has good storage stability.

[Brief description of drawings]

FIG. 1 is an optical micrograph (magnification about 700 times) of a stratum corneum sample obtained by the method of the present invention (Example-1).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/48 S 7638-2J A61B 5/10 300 Q

Claims (5)

[Claims] 1. A sticky substance which is soluble in an organic solvent is applied, and a tape to which the stratum corneum of the skin is adhered is attached to a transparent plate which has been applied with a sticking agent in advance. The method is characterized in that after immersing it in an organic solvent, the tape is removed to substantially fix a part of the stratum corneum on a transparent plate, and if necessary, this is dyed to prepare a stratum corneum sample. How to make a stratum corneum specimen. 2. The adhesive material is natural rubber or its derivative, SB.
R type rubber, NBR type rubber, butadiene-vinyl pyridine type rubber, butyl rubber, chloroprene type rubber, recycled rubber, cyanoacrylate type rubber, arabic rubber, tragacanth rubber, etc. The method for producing a stratum corneum sample according to claim 1, wherein: 3. The method for producing a stratum corneum sample according to claim 1, wherein the tape coated with an adhesive substance soluble in an organic solvent is cellophane tape. 4. The fixing agent is a single or a mixture of two or more selected from polyvinyl adhesives such as polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride and polyvinyl ether. The method for preparing a stratum corneum sample according to claim 1, wherein the method is contained in another base material. 5. The method for preparing a stratum corneum sample according to any one of claims 1 to 4, wherein the transparent plate is a glass plate.